JP2022184941A - Compositions containing decitabine, 5-azacitidine and tetrahydrouridine and uses thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide compositions for treating cancer and blood disorders, and to provide methods for treating the same.

SOLUTION: Compositions comprising decitabine and tetrahydrouridine or 5-azacytidine and tetrahydrouridine for the treatment of cancer are disclosed. The compositions in the form of a pill are administered to a human subject sequentially or alternately guided by the measurement of predictive biomarkers.

SELECTED DRAWING: None

COPYRIGHT: (C)2023,JPO&INPIT

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Nos. 62/262,839 filed December 3, 2015 and 62/429,292 filed December 2, 2016 .

This application relates generally to compositions containing decitabine (DEC), 5-azacytidine (5AZA) and tetrahydrouridine (THU) and methods of using the compositions for the treatment of cancer and hematological disorders in a subject. . Specifically, the composition in pill form comprises DEC and THU or 5AZA and THU, or a combination of DEC, 5AZA and THU, as a non-cytotoxic treatment for cancer or other hematologic disease in a subject.

Radiotherapy and drug therapy for cancer aim to use physiological pathways that can terminate cell growth and division. A specific physiological pathway that almost all anti-cancer drugs seek to use is the p53/p16-mediated apoptotic pathway (cytotoxicity). Since p53 and p16 are the two most commonly inactivated genes in cancer, this p53/p16 dependence of anticancer drugs and radiotherapy is the underlying principle of therapy resistance and relapse. Therefore, there is a need for new therapeutics that do not use the p53/p16 system to terminate cell proliferation and division of cancer cells.

Preclinical and clinical studies demonstrate that non-cytotoxic depletion of the enzyme DNA methyltransferase 1 (DNMT1) by decitabine (DEC) and 5-azacytidine (5AZA) is effective and does not require or depend on the p53/p16 system. (Negrotto S., et al. 2011 Cancer Res 71(4):1431-1441). However, 5AZA or DEC by themselves are not curative either in preclinical in vivo models of cancer or in clinical trials. Therefore, it is desirable to exploit mechanisms of cancer resistance to p53-independent therapy with 5AZA or DEC.

What this disclosure has identified is that cancer resistance to p53-independent therapy with 5AZA or DEC can be effectively overcome by logical, non-toxic and clinically applicable solutions. Specifically, the present disclosure provides compositions of non-toxic THU-DEC/THU-5AZA and methods and alternating regimens for overcoming cancer resistance by administration of compositions comprising non-toxic levels of THU-DEC. i.e., a method for administering the composition in a regimen that alternates intake of the composition, which may be in the form of pills or tablets containing THU-DEC and THU-5AZA, for a defined period of time. offer.

Thus, in one embodiment, an oral composition is provided for inducing safe, non-cytotoxic (p53/p16 independent) differentiation-mediated cell cycle exit of cancer cells.

Oral administration comprising a nucleoside analogue capable of irreversibly binding and depleting the enzyme DNA methyltransferase 1 (DNMT1), a potent cytidine deaminase (CDA) inhibitor and one or more pharmaceutically acceptable excipients wherein the nucleoside analogue drug and a potent cytidine deaminase inhibitor are sufficiently therapeutically effective to induce differentiation of refractory and resistant cancer cells without cytotoxicity to normal tissues. A composition that is present in an amount. A nucleoside analog drug that depletes the DNMT1 enzyme present in the composition is DEC. In one embodiment the nucleoside analogue drug may be 5AZA. The CDA inhibitor in the composition can be tetrahydrouridine (THU) or certain tetrahydrouridine derivatives such as 2'-fluorinated tetrahydrouridine derivatives. In this embodiment, when the nucleoside analogue drug that depletes DNMT1 is DEC, DEC may be present in an amount ranging from 1-10 mg/m ² . When the nucleoside analog drug present in the oral composition is 5AZA, 5AZA can be present in an amount ranging from 10-100 mg/m ² . The CDA inhibitor THU in oral compositions can be present in an amount ranging from about 400 mg/m ² to about 500 mg/m ² .

A therapeutic composition for inducing non-cytotoxic differentiation may be in the form of a pill or tablet or capsule. In one embodiment, a therapeutic composition may comprise the nucleoside analog drugs DEC and THU, and one or more pharmaceutically acceptable carriers. In another embodiment, a therapeutic composition may comprise 5AZA, THU and one or more pharmaceutically acceptable carriers. In some embodiments, a therapeutic composition may contain therapeutically effective amounts of both nucleoside analog drugs (DEC or 5AZA) combined with THU in a single pill.

A therapeutic composition may be administered to a subject after measurement of predictive biomarkers, including but not limited to DCK, in cancer cells. The development of predictive biomarkers can assist treating physicians in selecting appropriate treatment regimens so that treatment of particularly refractory and aggressive metastatic cancers in subjects is more effective. In this embodiment, the predictive biomarker may be deoxycytidine kinase (DCK), a cancer cell pyrimidine-metabolizing enzyme. The DCK enzyme is known to phosphorylate and sequester DEC in cells, and some cancer cells prevent DEC-induced DNMT-1 depletion by relying on the alternative pyrimidine-metabolizing enzyme UCK2, which is However, it makes cancer cells vulnerable to the drug 5-5AZA. Measurement of DCK in cancer cells before or during or after administration of the oral composition by functional molecular imaging by positron emission tomography (PET) utilizing DCK-dependent probes or by quantitative real-time (QRT)- It can be done by PCR.

In a therapeutic method for inducing non-cytotoxic differentiation of cancer cells in any mammalian tissue, a first therapeutic composition comprising DEC, THU and one or more pharmaceutically acceptable carriers, and A second therapeutic composition comprising 5-5AZA, THU and one or more pharmaceutically acceptable carriers is administered to a patient in need thereof, either sequentially, alternately, or simultaneously. obtain. In this embodiment, the tetrahydrouridine may also include derivatives such as 2'-fluorinated tetrahydrouridine derivatives. The first and second compositions may be administered after, during, or before measuring the prognostic biomarker in cancer cells.

In one embodiment, the first and second therapeutic compositions may each be in the form of a pill, or the compositions may be combined within a single pill. When the composition of the present disclosure is in the form of a pill, the THU or THU derivative may be located specifically on the surface of the pill, while DEC or 5AZA may be located inside the pill. Such differential positions of the nucleoside analogue drug, DEC or 5AZA, and THU on the pill are such that THU or a THU derivative is about 1 minute to 180 minutes or 15 minutes to about 180 minutes before either DEC or 5AZA. and more preferably 30 minutes to about 60 minutes prior to either DEC or 5AZA. In another embodiment, DEC or 5AZA may be polymer-coated or embedded in a polymer to facilitate delayed drug release compared to polymer-coated or polymer-embedded THU or THU derivatives. .

Upon oral administration of the therapeutic composition, the combination of DEC and THU achieved the oral bioavailability, low C _max and multiple hours required for depletion of non-cytotoxic DNMT-1 by DEC or 5AZA. At least about a 10-fold improvement in T _max can result. Due to the known off-target effects or toxic effects of DEC and 5AZA, optimizing DEC or 5AZA to deplete DNMT1 in vivo could have potent therapeutic effects. Thus, the therapeutic composition can provide peak plasma DEC levels of about 0.05 μm to about 0.5 μm, or peak plasma 5AZA levels of about 0.5 μm to about 5 μm in human subjects. In this embodiment, a patient with cancer first takes a pill containing DEC and THU, and days or weeks apart, takes a pill containing 5AZA and THU, or vice versa. As such, the treating physician may prescribe pills containing DEC and THU (or)-5AZA and THU in a continuous or intermittent fashion.

In one embodiment, methods are provided for treating any tumor, metastatic tumor or combination thereof in a subject's tissue, or hematological malignancies or hematological disorders in a subject. (i) administering a therapeutically effective amount of a first composition in the form of a pill comprising a first nucleoside analog drug, a cytidine deaminase inhibitor and a pharmaceutically acceptable carrier; administering a therapeutically effective amount of a second composition in the form of a pill comprising two nucleoside analog drugs, a cytidine deaminase inhibitor and a pharmaceutically acceptable carrier, wherein the first the nucleoside analogue drug in the composition of may be DEC, the nucleoside analogue drug in the second composition may be 5AZA, and the above first composition and the above second composition wherein the cytidine deaminase inhibitor in is THU or a THU derivative such as a 2'-fluorotetrahydrouridine derivative. In this embodiment, the first and second compositions may be administered sequentially, alternately, or simultaneously. Additionally, the first and second compositions can be administered prior to, during, or after measuring the level of the predictive biomarker in the subject's cancer cells.

Treatment selection depends on the determination of DCK levels in a patient in need of treatment and the appropriate nucleoside analogue drug (i.e., DEC and THU or 5AZA and THU in combination with THU in a manner that directs cancer tissue toward differentiation pathways). or both). The drug composition in the form of pills is prescribed once a week, or once every two weeks, or once to three times a week to patients suffering from cancer or hematological malignancies or blood diseases. good. In yet another embodiment, the composition in the form of a pill is administered to patients at risk of developing a hematological or solid malignancy, or previous history of a hematological or solid malignancy or hematological or hematologic disease. It may be prescribed between once every two weeks and three times a week in patients at risk of recurring diagnosis.

In one embodiment, a kit is provided for treating a tumor or refractory tumor or hematologic tumor or disease. The kit may comprise (i) a pill containing DEC, THU or THU derivative and carrier, (ii) a pill containing 5AZA, THU or THU derivative and carrier, and (ii) instructions. In another embodiment, the kit may include (i) a capsule containing a mixture of DEC/THU granules or microparticles and 5AZA/THU granules or microparticles, and (ii) instructions. The pill or capsule can be prepared so that the THU or THU derivative is always bioavailable at least 1 minute to about 60 minutes before the nucleoside analog drug, DEC or 5AZA. In one embodiment, the THU or THU derivative is bioavailable 1 minute to about 15 minutes, or 15 minutes to 30 minutes, 30 minutes to 45 minutes, 45 minutes to 60 minutes before DEC or 5AZA. can be

FIG. 2 is a graphical representation of CDKN2A and TP53 deletion/mutation prognosis in renal cell carcinoma (RCC). Graphical representation of amplified or deleted genes VHL, PBRM1 and Pax8 in RCC. FIG. 4 is a graph showing hypermethylated WT1 promoter CpGs in RCC compared to non-versus non-cancerous kidneys. Graphical representation of high expression of differentiation drivers and paradoxically epigenetic repression of key epithelial differentiation target genes (eg, WT1 and GATA3) in RCC. Comprehensive documentation showing Pax8 coactivator and corepressor interactions by LC/MSMS. Graphical representation of PAX8 interactions towards coactivators and corepressors resulting from DNMT1 depletion by non-cytotoxic concentrations of DEC. FIG. 4 is a graphical representation of DCK and UCK2 expression levels correlating with DEC and 5AZA sensitivity, respectively. Graphical representation of MDS/AML cells from Dec-treated patients with low DCK and high UCK2 levels and vice versa from 5-Aza-treated patients at relapse. FIG. 4 is a graphical representation of the effect of THU-DEC 3×/week alternating with THU-55AZA 3×/week in a xenograft model of patient-derived AML. FIG. 4 is a flow chart showing how biomarkers can guide rationale to extend response to drug and salvage relapse.

The present disclosure relates to compositions containing a nucleoside analogue drug and a pharmaceutically acceptable excipient in combination with a potent cytidine deaminase inhibitor. The nucleoside analogue drug present in the composition can be any known nucleoside deoxycytidine analogue drug that is also a DNA demethylating agent, including but not limited to DEC, 5AZA, gemcitabine, and the like.

The term "nucleoside analogue" refers to any nucleoside containing nucleic acid analogues and sugars that act as metabolic inhibitors when phosphorylated. Specifically, the nucleoside analogues of this disclosure refer to cytidine analogues, including but not limited to DEC with unmodified sugars and 5AZA. DEC (5-5-aza-2′-deoxycytidine) mimics the natural constituents of DNA, can irreversibly bind to and deplete the enzyme DNA methyltransferase 1 (DNMT1), and unmodified sugars. is relatively unique among the large family of nucleoside analogues in that it does not terminate DNA chain elongation and allows depletion of DNMT1 without cytotoxicity. In cancer cells, depletion of DNMT1 by the drug DEC prevents repression of differentiation genes and regenerates cancer cell differentiation—the aberrant proliferation of cancer cells is caused by a block in their normal differentiation process that is attenuated by DEC. be Furthermore, depletion of DNMT1 in normal stem cells increases their self-renewal, ie DNMT1 depletion increases the number of normal stem cells, opposite to its effect on cancer cells. Depletion of DNMT1 by DEC may therefore be an effective, extremely safe and well-tolerated cancer therapy.

In one embodiment of the present invention, the pharmacological goal of a non-cytotoxic treatment, defined as a treatment that does not induce cytotoxicity (apoptosis) of normally dividing cells, is the use of DEC or 5AZA to deplete DNMT1 in vivo. maximizing the time-above-threshold concentration (≧0.05-0.2 μM for DEC, ≧0.5-2 μM for 5AZA), while the high peak levels that damage DNA ( ≧0.5-1 μM for DEC and ≧5-10 μM for 5AZA). This exposure with a low C _max but prolonged T _max is also provided in solid tissues, where high CDA expression in solid tissues, including liver, kidney, intestine and brain, is the reason for the current formulations of DEC and 5AZA. distribution is negligible. The DNMT1-depleting effects of DEC or 5AZA in the present disclosure may be intermittent, as continuous exposure to DEC or 5AZA causes accumulation of DEC or 5AZA in normal cells to levels that induce cytotoxicity. intended. For example, currently known routes of administration, regimens and formulations of DEC or 5AZA can result in high peak levels of the drug, which cause cytotoxic killing of normal cells in sensitive tissues such as bone marrow. It provides a very short suprathreshold time concentration to deplete DNMT1 in bone marrow, especially except in solid tissues that highly express CDA. Also, currently known routes of administration, regimens, and formulations of DEC or 5AZA avoid continuous or sustained exposure that results in the accumulation of DEC or 5AZA in normal bone marrow cells to levels that cause cytotoxicity. Do not increase or distribute the exposure time to increase depletion of DNMT1. Instead, these current approaches mimic conventional regimens with conventional pulsatile dosing intended for cytotoxic therapeutic purposes. Importantly, disruption of DEC and 5AZA by the enzyme cytidine deaminase (CDA) resulted in a shortened half-life of <20 minutes in vivo (as opposed to a half-life of 5-9 hours in vitro) ( Liu, Z., et al. 2006 Rapid Common Mass Spectrum 20:1117-1126), this drastic reduction in half-life is a significant barrier to effective in vivo conversion of in vitro observations, especially at high levels of It is a barrier to solid tissue cancers that reside in tissues that endogenously express CDA. High peak levels of DEC or 5AZA in plasma and bone marrow are associated with pharmacogenomic alterations of CDA (Gilbert, JA, et al. 2006 Clin. Cancer Res 12, 1794-1803; Kirch, HC, et al. 1998 Exp. Hematol.26, 421-425; cause fluctuations. Also, malignant cells can develop resistance by destroying DEC with CDA (Ohta, T., et al. 2004 Oncol Rep 12:1115-1120; Hubek, I., et al. 2005 Br J Cancer 93 Huang, Y., et al. 2004 Cancer Res 64:4294-4301), and malignant cells may escape DEC therapeutic effects by being present in tissues with high levels of CDA (Ebrahem, Q. ., 2012 Oncotarget 3(10):1137-45).

To address these multiple fundamental pharmacological limitations of DEC and 5AZA, the oral route of administration of DEC or 5AZA in combination with the CDA inhibitor THU is preferred herein. Such oral administration of DEC or 5AZA in combination with THU substantially reduces peak levels compared to current parenteral administration, but substantially reduces concentrations for times above the threshold for depleting DNMT1. increase exponentially. Oral administration allows chronic, frequent, but not daily (i.e., regular) therapy to maintain life-long therapeutic efficacy, while dividing cells and preventing cytotoxicity: DEC or 5AZA can be distributed in solid tissue at the site and eliminate cancer cell havens in CDA-rich organs such as the liver. In short, this combination allows treatment of solid tissue cancers. Moreover, oral administration facilitates the widespread use of these drugs worldwide [028]. Cytidine deaminase (CDA) is an enzyme that is highly expressed in the liver and intestinal tract and rapidly destroys DEC and 5AZA in the body. In humans, the CDA gene is prone to non-synonymous single nucleotide polymorphisms that produce variants of cytidine deaminase that differ by more than 3-fold in enzymatic activity (Gilbert, JA, et al. 2006 Clin Cancer Res 12, 1794- Kirch, HC, et al., 1998 Exp Hematol 26, 421-425; Yue, L., et al., 2003 Pharmacogenetics 13, 29-38).

THU is known to inhibit cytidine deaminase or CDA and has many desirable properties, including that they exhibit a benign toxicity profile, well-characterized PK, intestinal and hepatic versus DEC and 5AZA oral bioavailability. producing more predictable effects of DEC or 5AZA drug doses from one individual to another; increasing concentrations of DEC or 5AZA over the threshold time to deplete DNMT1. , removing malignant cell refuges from DEC or 5AZA therapeutic effects, and the like. Accordingly, in one embodiment, the present invention provides a composition for oral administration, the composition comprising about 1-10 mg, with the CDA inhibitor THU in an amount ranging from about 400 mg/m ² to about 500 mg/m ² . /m ² of DEC, or 5AZA in amounts ranging from 10-100 mg/m ² . Although the CDA inhibitor used in the compositions of the present disclosure is THU, those skilled in the art will appreciate the fact that other CDA inhibitors such as Zebularine can be used in place of THU. In some other embodiments, the CDA inhibitor used in the therapeutic composition may be a commonly known THU derivative such as a 2'-fluorinated tetrahydrouridine derivative synthesized as an inhibitor of CDA. , Ferraris et al. 2014 J Med Chem. 57(6):2582-88, or in US Pat. No. 8,329,665 or EP2447272.

Malignant cells can develop resistance to DEC-THU combinations by shifting pyrimidine metabolism from salvage by DCK to salvage by UCK2, or by shifting pyrimidine metabolism from salvage by UCK2 to salvage by DCK. Since resistance to the 5AZA-THU combination can develop, it is conceivable that both drugs may be used alternately or sequentially to logically address the mechanisms of their respective resistance. In another embodiment, administration of the composition results in a plasma concentration of DEC from about 0.05 to about 0.5 μM or a concentration of 5AZA from about 0.5 to about 5 μM. In yet another embodiment, the composition is administered to a patient with metastatic cancer once a week or once every two weeks. In yet another embodiment, the composition is administered to a cancer patient 1-3 times per week. In some embodiments, the compositions of the present invention are useful in patients at risk of developing a hematological or solid malignancy, or at risk of recurrence of a previous diagnosis of a hematologic or solid malignancy. It is administered once every two weeks to three times a week.

5AZA integrates more extensively into RNA than DNA, and its integration results in suppression of protein production. When 5AZA integrates into DNA, it covalently binds DNA methyltransferases that interfere with DNA synthesis and subsequent cytotoxicity. In a preferred embodiment, DEC and 5AZA used in compositions for oral administration are in salt-free free drug form.

In one embodiment, the present disclosure provides a composition for oral administration in the form of a pill comprising THU and DEC or THU and -5AZA, wherein THU or a THU derivative (2'-fluorotetrahydrouridine derivative) is released more rapidly than DEC or 5AZA. Those skilled in the art will appreciate that such differential and/or dual release of drugs of the present disclosure may be achieved in any manner known in the art. "Differential release or dual release" refers to the release of the "drugs" at different times in a pill containing multiple drugs and/or the bioavailability of the drugs at different times after ingestion. For example, in differential release formulations, THU may be subject to faster dissolution rates, or THU may be located on the surface of the pill, while DEC or 5AZA are located inside the pill. THU or THU derivatives are bioavailable from about 1 minute to about 180 minutes prior to DEC or 5AZA, either due to location of THU or coating or embedding with a polymer or suitable vehicle; Alternatively, it is bioavailable from about 15 minutes to about 60 minutes prior to DEC or 5AZA, and alternatively from about 30 minutes to about 60 minutes prior to DEC or 5AZA. THU may be administered separately, with THU first followed by DEC or 5AZA in succession. As referred to herein, the term "bioavailable" refers to when an active agent (THU or DEC or 5AZA) can be absorbed by the body and used. "Orally bioavailable" refers to the ability of an active agent to be taken by mouth, absorbed into the body, and used.

In another embodiment, the composition for oral administration may be a single pill (also used herein as "capsules or tablets"), comprising THU or a THU derivative and at least one pharmaceutical drug. nucleoside analog drugs, both DEC and 5AZA, in addition to acceptable excipients. In this embodiment, DEC or 5AZA and THU are different on the surface of said pill such that THU or a THU derivative is bioavailable at least about 1 minute to about 180 minutes prior to DEC or 5AZA. located. In this embodiment, the drug DEC or 5AZA may be present in either coated or embedded granules or microparticles that facilitate release of the drug following THU or THU derivatives, respectively. This composition containing both cytidine analog drugs is administered to the patient for a defined period, such as once to three times a week for patients with aggressive and refractory cancer. In yet another embodiment, the composition is administered in patients at risk of developing a hematological or solid malignancy, or at risk of recurrence of a previous diagnosis of a hematologic or solid malignancy, for 2 weeks. 1 to 3 times a week.

The term "pharmaceutically acceptable excipient" as used in the composition is one that is compatible with (and preferably stabilizes) the active ingredients (e.g., compounds of the composition) of the pharmaceutical composition of the invention. can be used), and carriers and vehicles that are not harmful to the subject being treated. For example, solubilizers that form certain, more soluble complexes with the compounds of the present invention may be utilized as pharmaceutical excipients to deliver the compounds. Suitable carriers and vehicles are known to those skilled in the art. The term "excipient" as used herein includes all carriers, adjuvants, diluents, solvents or other inert additives and the like. Suitable pharmaceutically acceptable excipients include water, saline, alcohol, vegetable oils, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oils, fatty acid mono- and diglycerides, Including, but not limited to, petroesral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like. The pharmaceutical composition of the present invention may be sterilized and optionally contains auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers. , colorants, flavors and/or fragrances, etc., which do not adversely react with the active compounds of this invention.

In another embodiment, the composition of the invention is stored with a desiccant. This can help extend the shelf life of the compositions of the invention and facilitate their distribution and use on a global scale.

As used herein, the term "treating" refers to altering the disease course of the subject being treated. Therapeutic effects of treatment include, but are not limited to, preventing disease onset or recurrence, alleviating symptoms, reducing direct or indirect pathological consequences of disease, slowing disease progression, reversing or mitigating pathology; and remission or improved prognosis.

Pharmaceutical Compositions Pharmaceutical compositions of the present disclosure are disclosed in U.S. patent application Ser. /414,546 (issued as US Pat. No. 9,265,785). In one embodiment, the pharmaceutical compositions and non-toxic dosage forms of the present invention contain relative amounts of CDEC from about 0.1 to about 0.5 mg/kg or 5AZA from 1 to about 5 mg/kg and from about 10 to about 50 mg/kg. kg of THU and a pharmaceutically acceptable excipient, and a given pharmaceutical composition or dosage form releases inhibition of differentiation pathways in tumor cells, or Prepared to reduce abnormal suppression. A therapeutically effective amount and non-toxic dosage form of the pharmaceutical composition of the invention may exhibit or result in one or more of the following: (i) peak levels of DEC or 5AZA in the patient's plasma that are below the threshold; Above that threshold there is sufficient damage to DNA in normally dividing cells to cause apoptosis (cytotoxicity). (ii) an exposure time sufficient to deplete DNMT1 in cancer cells; It spans minutes to hours (5- to 20-fold extension of Tmax) (DEC or 5AZA alone have plasma exposure times of minutes), but it is still apoptotic (cytotoxic to DNA in normally dividing cells). below the threshold to accumulate intracellular levels of DEC or 5AZA to levels that induce sufficient damage to cause ). Briefly, optimized exposure time for non-cytotoxic depletion of DNMT1. (iii) a 5- to 20-fold increase in oral bioavailability of DEC or 5AZA; (iv) distribution of DEC or 5AZA into CDA-rich tissues where cancer cells could otherwise escape from the DEC or 5AZA therapeutic effects. This is the same principle underlying oral bioavailability, namely the ability of DEC or 5AZA to distribute in turn through the intestinal tract and liver. (v) induce expression of genes and proteins required for cell cycle exit by differentiation by cancer cells (p53/p16-independent cell cycle exit), and (v) act non-cellular to leave normally dividing cells. toxicity mechanism. Avoiding toxicity in this manner facilitates frequent intermittent therapy, increasing the proportion of cancers exposed to the effects of depleting DNMT1.

For example, in some embodiments, the pharmaceutical compositions and dosage forms of the present invention contain relative amounts of 1 to about 5 mg/kg 5AZA or 0.1 to about 0.5 mg/kg DEC and about 10 to about 50 mg /kg of THU as well as a pharmaceutically acceptable excipient, and a given pharmaceutical composition or dosage form releases inhibition of differentiation pathways in tumor cells or differentiation-associated genes in tumor cells. is formulated to reduce abnormal suppression of

The pharmaceutical composition or dosage form may contain additional active agents, including chemotherapeutic agents known in the art. The pharmaceutical composition may be administered alone or in combination with other known compositions to treat cancer in mammalian subjects. The term "in combination" can include simultaneous or sequential administration of the compositions of this disclosure. Sequential administration can also include administration of the therapeutic composition of the present disclosure followed by any known composition, or vice versa.

Administration of the composition may be carried out in single or multiple doses. Generally, the therapeutically active compounds of this disclosure are present in such dosage forms at concentration levels ranging from about 5.0% to about 70% by weight. In some embodiments, a subject in need of treatment for a tumor is administered a tablet or capsule containing 10 mg/kg THU and 0.2 mg/kg DEC or 2 mg/kg 5AZA twice weekly. or once a week or once every three days for about one or two weeks.

Sustained-release compositions in which the active ingredient is derivatized by differentially degradable coatings, e.g., microencapsulation, composite coatings, to enable differential release of THU or THU derivatives and nucleoside analog drugs, can be prepared. In some embodiments, nanoparticles of pharmaceutical compositions may be prepared as described in US Patent Application No. 14/046,343, which is incorporated herein in its entirety. These compositions ensure that THU is bioavailable from about 1 minute to about 180 minutes prior to DEC and/or -5AZA. In another embodiment, the micro- or nano-formulation ensures that THU is bioavailable from about 15 minutes to about 60 minutes, or from 30 minutes to about 60 minutes prior to DEC and/or -5AZA. do.

Compositions of the present disclosure may further contain other agents that reduce the rate by which the active compounds will decompose. Such agents include stabilizers including, but not limited to, antioxidants, ascorbic acid, pH buffers or salt buffers.

Predictive Biomarkers Developing “predictive or companion” biomarkers to guide treatment selection is a powerful and effective way to select meaningful individualized treatment regimens, such as dosages and schedules, for any subject in need thereof. It is a useful tool. Therefore, predictive biomarkers in the form of enzyme profiles are measured during treatment regimens to assist the treating physician in determining the appropriate treatment regimen for a subject. DNA synthesis and regeneration of genetic material are essential for cell proliferation, and high expression of the pyrimidine salvage enzyme DCK is a pan-cancer hallmark of proliferation. Therefore, radiolabeled nucleoside analogues can be used to image tumor growth via the salvage pathway of DNA synthesis. 1-(2′-deoxy-2′-[ ¹⁸ F]-fluoro-β-D-arabinofuranosyl)cytosine (TFAC) is a deoxycytidine analogue, such as DEC, which is one such radiotracer. is one. Since active DEC needs to be activated by DCK to produce its DNMT1-depleting effect, cancer DCK expression reliably predicts susceptibility to DEC across the spectrum of cancer histology and genetics. Therefore, TFAC-Positron Emission Tomography (PET) can be used to measure DCK-dependent cancer cell proliferation.

In some embodiments, predictive bioassays may be performed before, during, and after treatment with the disclosed compositions. This assay can be performed to select an appropriate therapeutic regimen. For example, if pre- or during-treatment assays show high FAC uptake that correlates with response to oral THU-DEC, the treating physician may continue to prescribe pills or tablets containing THU-DEC. Low or decreased FAC uptake associated with increased tumor size points to the predominance of cancer cells using the alternative pyrimidine-metabolizing enzyme UCK2, a mechanism of resistance to DEC, which is then investigated by physicians. may prescribe tablets or capsules containing THU-5AZA.

Critically, these measures do not simply provide scientific insight into the mechanisms of resistance, but guide the application of rational clinical measures to overcome resistance and prolong the response: (i) this The inability to achieve molecular PD with respect to the absence of side effects of cytotoxic therapy can lead to rational increases in dose or frequency of oral THU-DEC (Figure 9). (ii) resistance associated with loss of molecular PD and re-increase in burden of mutant allele frequency concomitant with shift in pyrimidine-metabolizing enzymes could lead to rational switch of therapy to oral THU-5AZA ( Figure 9). Optimal indications would probably incorporate oral THU-DEC administered chronically, chronically, alternating with oral THU-55AZA administered the following week (FIGS. 7-9).

Methods of Treatment In one embodiment, a pill containing a therapeutic composition of the present disclosure may be administered to a cancer patient in need of treatment. The cancer may be a solid or liquid cancer, or an aggressive and refractory cancer of any known tissue in the subject. Therapeutic compositions of the present disclosure may be used for hematologic malignancies or blood disorders such as, but not limited to, sickle cell anemia, hemoglobinopathies, thalassemia or US patent application Ser. No. 13/414,546 (now US patent 9,265,785, which is incorporated herein in its entirety). "Treatment or treating" refers to any known physical and/or biochemical and/or molecular alteration in cancerous tissue. For example, treatment may refer to amelioration of known cancer biomarker proteins, such as tumor size or levels of DNMT1 in normal and malignant cells, or derepression of specific differentiation genes or all of the above. , can be a molecule. Treatment methods include measuring predictive biomarkers before, during, or after treatment. Predictive biomarkers can inform the treating physician of the appropriate dosage and schedule to prescribe to the patient. For example, a patient may be administered a drug of the present disclosure as follows: oral THU 10 mg/kg, followed by oral DEC 0.2 mg/kg 60 minutes after THU, twice weekly on consecutive days. Treatment may include administering a pill containing THU-DEC or THU-5AZA, or a pill containing both DEC and 5-5AZA, wherein the THU or THU derivative is about 1% of DEC and/or 5AZA. minutes to about 180 minutes, or 15 minutes to about 180 minutes before.

Treatment regimens include administration of a first composition comprising DEC, THU or a THU derivative and at least one pharmaceutically acceptable excipient followed by 5AZA, THU or THU separated by days or weeks. Alternate administration of a second composition comprising the derivative and at least one pharmaceutically acceptable excipient may be included. This sequential or alternating treatment regimen may continue for weeks to months.

In some embodiments, the method of treatment includes antimetabolites, doxorubicin, HDAC inhibitors, antiviral agents, as described in detail in US Application No. 14/046,343, which is incorporated herein in its entirety. additional therapies including, but not limited to, radiotherapy or cytotoxic chemotherapeutic agents such as antiretroviral agents, antiretroviral agents, or any combination of these agents.

EXAMPLES The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1:
The need for therapeutics that are mechanistically different from current options Historically, most drugs evaluated in RCC attempted to terminate proliferation using the p53/p16 apoptotic system (cytotoxicity). For decades, such treatments have proven toxic and ineffective. This is because, like most cancer cells, RCC cells are apoptosis-resistant compared to normal cells due to genetic alterations in key p53/p16 system genes such as CDKN2A. The first drugs approved by the FDA for the treatment of RCC inhibited signaling pathways (eg, AKT/mTOR) that stabilized the MYC protein, a key coordinator of cell proliferation and division. Thus, these drugs interfere with RCC growth directly by affecting the MYC protein and indirectly by inhibiting the angiogenesis required to support RCC growth. Although such therapy is a clear advance, durable responses are rare (median survival 18-30 months), and changes in p53/p16 still influence response, suggesting that multivariate analyses. (Fig. 1). Moreover, second- or third-line responses were much less durable than first-line responses, suggesting some overlap in the mechanisms of resistance to the licensed drugs. It is therefore suggested and appropriate to evaluate mechanistically distinct therapeutic strategies that explicitly use pathways other than p53/pl6 to terminate RCC cell proliferation and division.

PBRM1 inactivation in RCC and epithelial differentiation therapy An unexpected finding from the RCC genomic genetic study is that genetic alterations in the coactivator subunits, particularly PBRM1 but also others (SETD2, SMARCC1, SMARCA2, ARID1A). It is essentially universally inactivating (Fig. 2). These findings are recent and the function of these coregulatory pathways in normal renal cells, or the pathways by which loss of function confers clonal advantage, was unclear. A possibly relevant finding was speculated to be the epigenetic repression of key epithelial terminal differentiation genes (eg GATA3, WT1) in RCC (Figs. 3, 4). This epigenetic repression of late differentiation genes was of particular interest. This is because RCC cells express unmutated master transcription factors that drive epithelial differentiation (eg PAX8, PAX2) at relatively conserved mRNA and high protein levels (Fig. 4). Another relevant finding was the correlation of increased chromatin suppression marks with poor prognosis in multiple RCC studies. In the final funding cycle, we empirically correlated these findings, suggesting that PBRM1/BAF is a major driver of mesenchymal epithelial transition 11-16, which is highly expressed in RCC17-22, transcriptional activation by PAX8. was found to mediate PBRM1 loss causes disproportionate recruitment of corepressor counterparts to PBRM1/BAF (eg, DNMT1) to PAX8, repressing instead of activating epithelial differentiation target genes that terminate proliferation (FIG. 5). Importantly, inhibiting these specific co-repressors (e.g., DNMT1) restores expression of multiple PAX8 target genes, regenerating epithelial differentiation and physiological, p53/p16-independent cell cycle exit. (Fig. 6).

Example 2:
Need for Mechanism-Based Predictive Biomarkers DNA synthesis and regeneration of genetic material are essential for cell proliferation, and high expression of the pyrimidine salvage enzyme DCK is a hallmark of pan-cancer growth. Radiolabeled nucleoside analogues have therefore been developed to image tumor growth via the salvage pathway of DNA synthesis. 1-(2'-deoxy-2'-[18F]-fluoro-β-D-arabinofuranosyl)cytosine (FAC) is one such radiotracer, as is the deoxycytidine analogue Dec. be. However, it was stated that, like Dec, FAC did not distribute properly in solid tissues such as liver and kidney due to rapid deamination by CDA. Co-injection of THU-FAC (TFAC) then resolved this issue, leading to high FAC uptake in hepatocellular carcinoma versus background liver in a woodchuck animal model of viral infection that induced primary hepatocellular carcinoma. turned out to bring about As shown in FIG. 10, the use of predictive biomarker assays helps determine whether the frequency or dosage of THU-Dec needs to be increased.

Thus, TFAC-PET could suggest a rational salvage therapy. If validated, it may also be involved in prior treatment selection between THU-Dec versus THU-5Aza. Furthermore, it is possible that TFAC-PET captures an aspect of RCC biology that correlates well with response and outcome to other RCC drugs (such as sunitinib). In other words, it is conceivable that TFAC-PET could fulfill the role of broad predictive biomarker that FDG-PET could not. The potential utility demonstrated herein may justify evaluation for such broad purposes.

Example 3:
Refractory malignant cells in DEC-treated patients increase UCK2 and decrease DCK, and vice versa in 5AZA-treated patients. Patients were harvested before and at relapse, and the expression of the pyrimidine-metabolizing enzymes DCK and UCK2 was measured by quantitative real-time PCR. As shown in FIG. 8, during relapse, MDS/AML cells from Dec-treated patients had lower DCK and higher UCK2, and the opposite in 5Aza-treated patients.

Example 4:
Non-cytotoxic treatment with alternating THU-DEC at 3X/week and THU-5AZA at 3X/week reduced response compared to DEC alone or THU-DEC alone in a xenograft model of patient-derived AML. Primary AML cells (1×10 ⁶ cells) from the bone marrow of a significantly prolonging AML patient were xenografted into NSG mice. Five days after inoculation, mice received vehicle, DEC alone at 0.2 mg/kg subcutaneously 3X/week, THU-DEC (THU 8 mg/kg and DEC 0.1 mg/kg) subcutaneously 3X/week, or THU. - Treated with THU-DEC 3X/week alternating with 5AZA 3X/week (THU 8 mg/kg and 5AZA 1 mg/kg). n=5 per treatment group. As can be seen from Figure 9, alternating regimens of THU-Dec and THU-5Aza significantly increase the survival probability in these xenograft models of patient-derived AML.

Claims (20)

5-azacytidine,
Tetrahydrouridine, and a pharmaceutically acceptable excipient,
A composition for oral administration comprising
A composition comprising from about 10 to about 100 mg/m ² of 5-azacytidine and from about 400 to 500 mg/m ² of tetrahydrouridine. 2. The composition of claim 1, wherein said composition is effective to produce a peak plasma concentration of 5-azacytidine of about 0.5 μM to about 5 μM. 3. The composition of claim 2, wherein said composition is effective to produce a peak plasma concentration of 5-azacytidine of from about 0.5 μM to about 2 μM. 2. The composition of claim 1, wherein the tetrahydrouridine in the composition is a 2'-fluorinated tetrahydrouridine derivative and is bioavailable from about 1 minute to about 180 minutes before 5-azacytidine. 2. The composition of claim 1, wherein said composition further comprises decitabine in an amount ranging from about 1 mg/ ^m2 to about 10 mg/ ^m2 . 2. The composition of claim 1, wherein said composition is in the form of a pill or tablet or capsule. 2. The composition of claim 1, wherein said composition is effective in treating cancer or a blood disorder in a subject. A method for treating cancer or a blood disorder in a subject, comprising:
administering to said subject a first composition comprising decitabine, tetrahydrouridine and a pharmaceutically acceptable carrier;
administering to said subject a second composition comprising 5-azacytidine, tetrahydrouridine and a pharmaceutically acceptable carrier;
including
The method wherein said first and said second compositions are administered to said subject sequentially, alternately or simultaneously. 9. The method of claim 8, wherein the first composition administered to the subject comprises about 1 to about 10 mg/ ^m2 of decitabine and about 400-500 mg/ ^m2 of tetrahydrouridine. 9. The method of claim 8, wherein the second composition administered to the subject comprises about 10 to about 100 mg/ ^m2 of 5-azacytidine and about 400-500 mg/ ^m2 of tetrahydrouridine. 9. The method of claim 8, wherein administration of said first and said second composition results in a peak decitabine plasma concentration of less than about 0.5 μm and a peak 5-azacytidine plasma concentration of at most 5 μm in said subject. 9. The method of claim 8, wherein tetrahydrouridine in said first and said second compositions is bioavailable from about 1 minute to about 180 minutes before decitabine or 5-azacytidine. the tetrahydrouridine in the first and second compositions is a 2'-fluorinated tetrahydrouridine derivative and is bioavailable from about 15 minutes to about 180 minutes before decitabine or 5-azacytidine; 9. The method of claim 8. 9. The method of claim 8, wherein the first composition is present in a first pill and the second composition is present in a second pill. 9. The method of claim 8, wherein said cancer comprises benign, metastatic or refractory cancer of any tissue. 9. The method of claim 8, wherein said blood disease comprises sickle cell disease, thalassemia, or hemoglobinopathy. A method of treating cancer in a subject comprising:
administering a first composition comprising decitabine, tetrahydrouridine and a pharmaceutically acceptable carrier;
administering a second composition comprising 5-azacytidine, tetrahydrouridine and a pharmaceutically acceptable carrier;
including
A method, wherein said first and said second compositions are administered after measuring a predictive biomarker in said subject. 18. The method of claim 17, wherein the predictive biomarker measurement comprises the level of deoxycytidine kinase in the subject. 18. The method of claim 8 or 17, wherein said subject is a mammal. A dual release pill for treating cancer or a blood disorder in said subject comprising decitabine, tetrahydrouridine and at least one pharmaceutically acceptable excipient, wherein said decitabine and said tetrahydrouridine A dual release pill separately located on the pill surface to facilitate the release of each drug at different times when orally ingested by the subject.

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